One of the major foci of research in the Laboratory for Integrative Neuroscience (LIN), Section on Synaptic Pharmacology, is the determination of mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. In the preceding year we found that ethanol, at low concentrations associated with mild intoxication, altered two forms of synaptic plasticity in the dorsal striatum. At synapses between cortical and striatal neurons, ethanol inhibited long-term potentiation (LTP) a long-lasting increase in synaptic function, while enhancing the expression of long-term depression (LTD) a long-lasting decrease in synaptic function. We have thus continued our examination of the mechanisms involved in striatal LTD with an eye to identifying potential sites of ethanol interaction. We know that striatal LTD involves a decrease in presynaptic glutamate release probability brought about, at least in part, by activation of presynaptic cannabiniod type 1 (CB1) receptors. Activation of D2-type dopamine receptors, metabotropic glutamate receptors, and L-type calcium channels are also implicated in the induction of LTD. However, it has not been clear how these different molecules participate in this form of plasticity. We have used a direct activator of L-type channels to induce LTD, and the plasticity activated under this condition is independent of D2 and metabotropic glutamate receptors. We believe that the L-type channel is a critical molecular switch for LTD induction, most likely acting to supply the major source of intracellular calcium necessary for production of the endogenous cannabinoids that act as retrograde messengers and stimulate CB1 receptors invovlved in LTD induction.[unreadable] Our studies also indicate that postsynaptic release of endogenous cannabiniods (endocannabinoids) involves a synaptically-regulated step. In electrophysiological experiments we use a micropipette to fill the postsynaptic striatal neuron with the endocannabinoid. When this loading is combined with moderate levels of synaptic activation, we observe a slowly-developing synaptic depression that is blocked by CB1 receptor antagonist. If, however, stimulation is given at low frequencies no synaptic depression is observed. Furthermore, inclusion of a blocker of the putative endocannabinoid membrane transporter in the postsynaptic cell blocks the synaptic depression. These findings indicate that endocannabinoid release from the postsynaptic cell is driven, at least in part, by synaptic activation. The regulated release step appears to involve a membrane transport or carrier system, but is independent of intracellular calcium and glutamate receptor activation. This mechanism is one potential target for alcohol actions in striatum.[unreadable] Our studies in previous years have focused on LTD at excitatory, glutamatergic synapses in striatum. However, in the course of these latest studies we also observed that LTD can also be induced at inhibitory synapses in striatum that use the neurotransmitter gamma-aminobutyric acid (GABA). Our most recent findings indicate that the striatal GABAergic synapses are much more sensitive than glutamatergic synapses to CB1 activation-induced synaptic depression and LTD. These findings have important implications for our understanding of the role of CB1 receptors and the impact of cannabinoid drugs on striatal circuitry. It will be necessary to determine the different roles of cannabiniod and endocannabinoid actions on excitatory and inhibitory striatal synapses in striatal function and striatal-based learning and memory (including skill and habit learning). In future studies, we hope to examine genetically engineered mice that lack CB1 receptors in different neurons within the corticostriatal circuitry in order to determine the cellular locus of CB1 receptors that participate in different aspects of striatal-based learning and memory, as well as development of habitual behavior that contributes to addiction.[unreadable] These several lines of research should allow us to gain a better understanding of synaptic mechanisms of learning and addiction. In addition, we are obtaining further evidence of the mechanisms through which alcohol can alter neuronal mechanisms involved in learning and memory in brain regions implicated in addiction.[unreadable] Another long-standing interest within our laboratory and section is the molecular basis of ethanol actions on the brain. In this contex, we have studied the 5-HT3 receptor for the neurotransmitter 5-hydroxytryptamine (aka serotonin), a protein that is functionally altered by pharmacologically-relevant concentrations of ethanol. One of our goals has been to understand the relationship between 5-HT3 structure, function and pharmacology. As a first step in this direction we have been working with Dr. Tina Machu of the North Texas Health Sciences University and Dr. Michael Blanton of the Texas Tech Health Sciences University to purify this protein and begin to use biophysical techniques to probe 5-HT3 structure. We developed a receptor with an amino acid pharmatope tag based on the structure of the alpha-bungarotoxin binding motif on the nicotinic acetylcholine receptor. We also created stable cell lines expressing this pharmatope-tagged receptor. Using these stable lines and a bungarotoxin affinity purification scheme, Dr. Blanton has been able to obtain a sufficient amount of purified receptor to perform photoaffinity labeling with a probe that recognizes sites of protein-lipid interaction within the membrane. This probe specifically labels a few sites within the membrane-spanning domains of the 5-HT3 protein. In future studies we hope to examine effects of other probes, such as general anesthetics, and we also hope to purify sufficient quantities of the receptor to perform additional biophysical analyses that will help us to better understand receptor function. Ultimately, we hope to use these techniques to determine how ethanol alters receptor structure and perhaps develop molecular techniques to interfere with these alcohol actions.